Stringent positive and negative control of the proliferative expansion of mesenchymal cells, osteoprogenitor cells and immature osteoblasts is critical for normal skeletal development and bone formation. Formation of a mineralized skeleton requires the runt-related transcription factor RUNX2/CBFA1, but new evidence indicates that its biological function extends beyond that in osteoblast maturation. Our central hypothesis is that RUNX2/CBFA1 is a principal regulator of osteoblast proliferation. This hypothesis will be addressed by investigating three related questions. First, does inactivation of the RUNX2 protein genetically influence cell growth parameters in osteoprogenitors (Aim 1)? The proposed studies will examine whether inactivation of RUNX2 alters cell growth characteristics of RUNX2 deficient calvarial cells, and whether re-introduction of RUNX2 restores normal cell growth control. Second, which transcriptional co-regulator of RUNX2 mediates its osteoblast proliferation-related biological functions (Aim 2)? RUNX2 interacts with several distinct co-factors that modulate its gene regulatory activity. To address this question, the critical co-regulatory factor that together with RUNX2 mediates bone cell growth regulation will be characterized. Third, which RUNX2 regulated genes contribute to growth control of osteogenic cells (Aim 3)? This question will be addressed by identifying downstream target genes of RUNX2 that together with RUNX2 mediate the proliferation-related regulatory properties of RUNX2. The results of these studies will provide key insights into the RUNX2 dependent molecular mechanisms and gene regulatory programs that control osteoblast proliferation. Understanding the mechanisms by which growth of osseous cells is controlled may permit manipulation of their cell growth potential for clinical applications in the treatment of skeletal diseases, bone fractures and/or bone-directed gene therapy.